This invention relates to delivery of a bioactive agent. More particularly, the invention relates to a composition and method of use thereof for delivering bioactive agents, such as DNA, RNA, oligonucleotides, proteins, peptides, and drugs, to an individual in need thereof.
The concept of using polymers for the controlled release of active drugs and other therapeutic compounds for medical applications has emerged and developed extensively in the last two decades.
When polymers are used for delivery of pharmacologically active agents in vivo, it is essential that the polymers themselves be nontoxic and that they degrade into non-toxic degradation products as the polymer is eroded by the body fluids. Many synthetic biodegradable polymers, however, yield oligomers and monomers upon erosion in vivo that adversely interact with the surrounding tissue. D. F. Williams, 17 J. Mater. Sci. 1233 (1982). To minimize the toxicity of the intact polymer carrier and its degradation products, polymers have been designed based on naturally occurring metabolites. Probably the most extensively studied examples of such polymers are the polyesters derived from lactic or glycolic acid and polyamides derived from amino acids.
A number of bioerodable or biodegradable polymers are known and used for controlled release of pharmaceuticals. Such polymers are described in, for example, U.S. Pat. No. 4,291,013; U.S. Pat. No. 4,347,234; U.S. Pat. No. 4,525,495; U.S. Pat. No. 4,570,629; U.S. Pat. No. 4,572,832; U.S. Pat. No. 4,587,268; U.S. Pat. No. 4,638,045; U.S. Pat. No. 4,675,381; U.S. Pat. No. 4,745,160; and U.S. Pat. No. 5,219,980. Of particular interest is U.S. Pat. No. 5,219,980, which describes ester bonds with side chains of amino-methyl or amino-ethyl groups. The products of hydrolysis of such compounds include 4-amino-2-hydroxy butanoic acid and 4-amino-3-hydroxy butanoic acid, which are not precursors for the twenty naturally occurring alpha-amino acids, and are, therefore, not as fully biocompatible as might be desired.
The biodegradable polymers, polylactic acid, polyglycolic acid, and polylactic-glycolic acid copolymer (PLGA), have been investigated extensively for nanoparticle formulation. These polymers are polyesters that, upon implantation in the body, undergo simple hydrolysis. The products of such hydrolysis are biologically compatible and metabolizable moieties (i.e. lactic acid and glycolic acid), which are eventually removed from the body by the citric acid cycle. Polymer biodegradation products are formed at a very slow rate, hence do not affect normal cell function. Drug release from these polymers occurs by two mechanisms. First, diffusion results in the release of the drug molecules from the implant surface. Second, subsequent release occurs by the cleavage of the polymer backbone, defined as bulk erosion. Several implant studies with these polymers have proven safe in drug delivery applications, used in the form of matrices, microspheres, bone implant materials, surgical sutures, and also in contraceptive applications for long-term effects. These polymers are also used as graft materials for artificial organs, and recently as basement membranes in tissue engineering investigations. 2 Nature Med. 824-826 (1996). Thus, these polymers have been time-tested in various applications and proven safe for human use. Most importantly, these polymers are FDA-approved for human use.
Nanoparticles are hypothesized to have enhanced interfacial cellular uptake because of their sub-cellular size, achieving in a true sense a "local pharmacological drug effect." It is also hypothesized that there would be enhanced cellular uptake of drugs in nanoparticles (due to endocytosis) compared to the corresponding free drugs. Several investigators have demonstrated that nanoparticle-entrapped agents have higher cellular uptake by endocytosis and prolonged retention compared to the free drugs. Thus, nanoparticle-entrapped drugs have enhanced and sustained concentrations inside cells and hence enhanced therapeutic drug effects in inhibiting proliferative response. Furthermore, nanoparticle-entrapped drugs are protected from metabolic inactivation before reaching the target site, as often happens with upon the systemic administration of free drugs. Therefore, the effective local nanoparticle dose required for the local pharmacologic drug effect may be several fold lower than with systemic or oral doses.
Nanoparticles have been investigated as drug carrier systems in cancer therapy for tumor localization of therapeutic agents, for intracellular targeting (antiviral or antibacterial agents), for targeting to the reticuloendothelial system (parasitic infections), as an immunological adjuvant (by oral and subcutaneous routes), for ocular delivery for sustained drug action, and for prolonged systemic drug therapy. 263 Science 1600-1603 (1994).
Because the surfaces of both nanoparticles (or microspheres) and cell membranes are negatively charged, cellular uptake is very low. Nanoparticles and microspheres of PLGA are electrostatically repelled by cell membranes, and thus cannot efficiently penetrate cells.
Since the early efforts to identify methods for delivery of nucleic acids in tissue culture cells in the mid 1950's, H. E. Alexander et al., 5 Virology 172-173 (1958), steady progress has been made toward improving delivery of functional DNA, RNA, and antisense oligonucleotides in vitro and in vivo. Delivery and expression of nucleic acids is a topic that continues to capture scientific attention. Methods for delivering functional non-replicating plasmids in vivo are currently in their infancy, while some success has been achieved in vitro. Current transfection techniques including using cationic lipids, E. R. Lee et al., 7 Human Gene Therapy 1701-1717 (1996), cationic polymers, B. A. Demeneix et al., 7 Human Gene Therapy 1947-1954 (1996); A. V. Kabanov et al., 6 Bioconjugate Chem. 7-20 (1995); E. Wagner, 88 Proc. Nat'l Acad. Sci. USA 4255-4259 (1991), viral vectors, A. H. Jobe et al., 7 Human Gene Therapy 697-704 (1996); J. Gauldie, 6 Curr. Opinion Biotech. 590-595 (1995). Each of the above listed methods has specific disadvantages and limitations. Viral vectors have shown a high transfection efficiency compared to the non-viral vectors, but their use in vivo is severely limited due to several drawbacks, such as targeting only dividing cells, random DNA insertion, risk of replication, and possible host immune reaction. J. M. Wilson et al., 96 J. Clin. Invest. 2547-2554 (1995).
Compared to viral vectors, nonviral vectors are easy to make and less likely to produce immune reactions, and there is no replication reaction. Under some conditions transfection efficiencies close to 100% can be obtained in vitro. In general, however, such nonviral vectors have been found to be ineffective for the introduction of genetic material into cells, and exhibit relatively low gene expression in vivo. For example, various cationic amphiphiles have been used for gene transfection. F. D. Ledley, 6 Human Gene Therapy 1129-1144 (1995). Transfection efficiency using cationic lipids, however, is still not as high as with viral vectors, and there have been complaints of cytotoxicity. The biggest disadvantage of cationic lipids is that they are not metabolites of the body and thus are very difficult to remove therefrom.
Several different classes of cationic polymers have been described for enhancing the uptake of DNA into cells and its egress from endosomes. Dendrimers, are polyamidoamine cascade polymers wherein the diameter is determined by the number of synthetic steps. Dendrimer-DNA complexes have been constructed using dendrimers of different size as well as different drug charge ratios (cationic dendrimer to anionic DNA). These complexes exhibit efficient gene delivery into a variety of cell types in vitro. F. D. Ledley, 6 Human Gene Therapy 1129-1144 (1995). Another type of cationic polymers, polyethylenimine (PI) and poly-L-lysine (PLL), similarly have high uniform positive charge density, will complex with DNA and other nucleic acids, and will transfer nucleic acids into a variety of cells in vitro. These polymers are capable of condensing plasmid DNA to form complexes with varying sizes and charges that may interact with the membrane of cells by ionic interaction and enter cells by endocytosis. These cationic polymers, however, are not biodegradable. Therefore, they are toxic due to accumulation in the body. It takes a few years, for example, to completely degrade PLL in the body.
In view of the foregoing, it will be appreciated that providing a carrier that is non-toxic, biodegradable, and efficient for delivery of nucleic acids and other drugs would be a significant advancement in the art.